The major aim of this project is to elucidate the mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that those cells expressing useless or self-reactive receptors do not mature (positive and negative selection). Both of these selection processes require TCR engagement of self-MHC antigens, but different aspects of these interactions determine whether the cells will live or die. Thymocytes also receive signals that promote their maturation and direct them into specific lineages. Precursors first must specify the alpha-beta or gamma-delta T cell fate. Those that develop in the alpha- beta pathway must then choose whether to become CD4 helper or CD8 cytotoxic T cells. Thus, our primary focus has been on the role of the T cell antigen receptor (TCR) and how it works together with other developmental cues to specify cell fate. In our previous work, we found that the type of TCR in the initial lineage choice (gamma-delta vs. alpha-beta) or the involvement of the coreceptors, CD4 or CD8, in the second decision (CD4 vs. CD8 lineage) could impose a bias on lineage choice. Moreover, we found that signals through the highly conserved transmembrane receptor, Notch, also influences these cell fate decisions. A constitutively active form of Notch can override the bias normally imposed by specific TCR signals, suggesting that the two signaling systems may act in concert to specify cell fate. Thus, we are investigating both TCR and Notch in developing thymocytes to determine how they are interrelated. We find that the expression of the TCR- associated signal transducing proteins, Syk and ZAP-70 are developmentally regulated in fetal thymocytes such that Syk is expressed prior to ZAP-70. This result suggests a model for regulating the gamma-delta vs. alpha-beta T lineage decision that is based on the developmental status of the TCR signaling apparatus rather than the specific isotype of the TCR. Our studies of TCR transgenic and gene- targeted mutant mice indicate that quantitative differences in TCR signaling determine whether a thymocyte will undergo positive selection, negative selection, or fail to be selected at all. Moreover, the quantity of signal within the acceptable range for positive selection also influences whether the immature T cell will become a CD4 or CD8 T cell. Quantitative differences in TCR signaling affect the gamma-delta vs. alpha-beta T lineage choice as well. - development, transgene, thymus, lineage, T cells, TCR, mouse, Notch